Mechanistic aspects of the cytotoxic activity of glufosfamide, a new tumour therapeutic agent

Abstract

β- D -Glucosyl-ifosfamide mustard (D 19575, glc-IPM, INN = glufosfamide) is a new agent for cancer chemotherapy. Its mode of action, which is only partly understood, was investigated at the DNA level. In the breast carcinoma cell line MCF7 glufosfamide inhibited both the synthesis of DNA and protein in a dose-dependent manner, as shown by the decreased incorporation of [3H-methyl]-thymidine into DNA and [14C]-methionine into protein of these cells. Treatment of MCF7 cells with 50 μM glufosfamide was sufficient to trigger poly(ADP-ribose) polymerase (PARP) activation, as revealed by immunofluorescence analysis. Both CHO-9 cells, which are O6-methylguanine-DNA methyltransferase (MGMT)-deficient, and an isogenic derivative, which has a high level of MGMT, showed the same cytotoxic response to β- D -glc-IPM, indicating that the O6position of guanine is not the critical target for cytotoxicity. By contrast, a sharp decrease in survival of cross-link repair deficient CL-V5 B cells was observed already at concentrations of 0.1 m Mβ- D -glc-IPM, whereas the wild-type V79 cells showed a 90% reduction in survival only after treatment with 0.5 m M of this compound. The therapeutically inactive β- L -enantiomer of glufosfamide also showed genotoxic effects in the same assays but at much higher doses. This was probably due to small amounts of ifosfamide mustard formed under the conditions of incubation. The results indicate that the DNA crosslinks are the most critical cytotoxic lesions induced by β- D -glc-IPM. © 2000 Cancer Research Campaign

Induction of DNA breaks and apoptosis in crosslink-hypersensitive V79 cells by the cytostatic drug β-D-glucosyl-ifosfamide mustard

“…The cell line CL-V5B is *30-fold more sensitive (based on D 10 values) than the parental 'wild-type' cell line to the cytotoxic effect of b-D-Glc-IPM, as measured by loss of colony-forming ability of the cells (Seker et al, 2000 and Figure 1). To see whether this hypersensitivity is due to the induction of apoptosis and/or necrosis, both endpoints were measured in parallel by annexin V and propidium iodide double-staining and flow cytometry as previously described (Ochs and Kaina, 2000).…”

“…Once inside the cell the drug is cleaved preferentially by intracellular glucosidases to form the ultimate cytostatic drug ifosfamide mustard (Seker et al, 1996). It has previously been shown that treatment of MCF-7 cells with b-D-Glc-IPM leads to activation of poly(ADP-ribose) polymerase (Seker et al, 2000). In the same study, by using an in vivo model system consisting of isogenic cell lines deficient and proficient for either O 6 -methylguanine-DNA methyltransferase (MGMT) or crosslink repair, it could be demonstrated that the most critical cytotoxic lesions caused by b-D-Glc-IPM are very likely to be DNA crosslinks (Seker et al, 2000).…”

“…Since this drug causes cytotoxicity very likely by inducing DNA crosslinks (Seker et al, 2000), parameters determining cell death were examined by comparing the response of cells that are hypersensitive to crosslinking agents (CL-V5B cells) with the corresponding parental cell line (V79). We show that CL-5B cells respond with a much higher frequency of apoptosis than the parental cells.…”

Glycolysis inhibition for anticancer treatment

“…This compound has been tested for its therapeutic activity against head and neck cancer, pancreatic adenocarcinoma, and non-small-cell lung cancer (Briasoulis et al, 2003;Dollner et al, 2004;Giaccone et al, 2004). Glufosfamide represent a novel oxazaphosphorine analog that uses the glucose transporter system for cellular entry to damage nuclear DNA (Seker et al, 2000).…”

Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences

“…Using an immuno chemical approach, Rosenthal et al (1998) Hinshaw et al (1999) analyzed PAR formation in permeabilized endothelial cells and keratinocytes upon treatment with 250 and 500 mM of SM for 2, 3, and 5 h, showing a 2 4 fold increase in signal intensities (Hinshaw et al, 1999). Using fluorescence microscopy, Seker et al (2000) showed PAR formation in MCF7 cells 24 h after glufosfamide treatment (50 750 mM). Using biotinylated NAD + in an ELISA approach, Kehe et al assessed PAR formation in permeabilzed HaCaT cells upon pulse treatment of cells for 30 min with 10 1000 mM SM followed by further incubation of cells for 45 min in SM free medium leading to a maximum four fold increase in signal intensities (Kehe et al, 2008).…”